During the course of spontaneous resolution of acute inflammation, ω-3 fatty acids are precursors for the biosynthesis of newly uncovered anti-inflammatory and pro-resolving lipid mediators (1
). These new families of mediators, coined resolvins and protectins, were identified in vivo
in resolving exudates and serve in molecular circuits that actively promote endogenous anti-inflammation and resolution of local inflammation (35
). However, the mechanism of ω-3 fatty acid mobilization in vivo
during inflammation-resolution has not been addressed. In this present report, evidence for new mechanisms is presented which indicates that unesterified ω-3 fatty acids also known as “free” fatty acids rapidly appear within the inflammatory exudates moving directly from circulation into the site of inflammation. The movement of EPA and DHA parallels those of both albumin and trafficking leukocytes. Also, single cell analyses of human PMN using a newly engineered microfluidics chamber provide direct evidence that DHA-derived resolvin D1 at nanomolar concentrations, and not its precursor DHA at equimolar levels, stops PMN chemotactic responses to spatial gradients of the chemokine IL-8.
Once formed, resolvins are active on target cells in their immediate milieu and are then inactivated by site-specific metabolism (26
). To enhance and prolong their actions, analogs of both RvD1 and RvE1 were prepared that delay their local inactivation in tissues, which proved to protect organs in vivo
from ischemia-reperfusion injury by reducing neutrophil tissue infiltration. These results demonstrate for the first time that ω-3 levels in circulating blood are rapidly made available to sites of inflammation in vivo
for their local utilization by exudates to generate potent bioactive local mediators, i.e., resolvins in situ
. The resolvins in turn act directly on target cells to stimulate endogenous anti-inflammation and protect organs from excessive neutrophil infiltration and subsequent local tissue damage.
After ingestion, EPA and DHA are distributed throughout the human body (41
). DHA is predominantly distributed in retina, sperm, cerebral cortex, spleen and red blood cells, whereas EPA is found in muscle, liver, spleen and red blood cells (42
). For example, DHA is esterified in phospholipids of microglial cells in culture and on activation of these cell, DHA is released from the phospholipids for enzymatic processing (43
). DHA is also, from recent results, the precursor for two separate families of mediators that are structurally distinct, namely D series resolvins and protectins. The protectins possess potent biological actions and a conjugated triene structure as distinguishing features (19
). EPA is the precursor for E series resolvins that show potent actions in several complex disease models, including inflammatory bowel disease, periodontal diseases and asthma (reviewed in ref. (45)
). However, the availability of unesterified or “free” ω-3 fatty acids EPA and DHA for processing during inflammation-resolution was of interest in the present studies and required a multidisciplinary approach and new tools to address these key points.
The level of total fatty acids in human blood is ~343 mg/100 ml plasma (46
). Based on this value and Suppl. Table 1
, between 48 to 490 mg of EPA and DHA exist in human blood as basal levels. The contribution of de novo
ω-3 fatty acids to the total amount in healthy human subjects, however, appears to be quite low. The proportion of a-linolenic acid converted to EPA is likely on the order of 0.20 to 8.0%, and the extent of conversion of α-linolenic acid to DHA is 0.05 to 4.0 % (47
). Thus, humans require ω-3 fatty acid intake via diet and/or supplementation. Currently, the FDA states that the dietary intake of EPA and DHA should not exceed 3 g/day (49
) since excess supplementation on the order of a gram/day was found to reduce the already low endogenous EPA and DHA biosynthesis (47
Although DHA and EPA are widely believed to possess anti-inflammatory properties themselves, the specific mechanisms responsible for these actions are still evolving. The ω-3 fatty acids are generally thought to replace the sn-2 position in phospholipid stores that is usually the positional site of esterified ω-6 fatty acids such as arachidonic acid (41
). It is well appreciated that, upon activation, cells release arachidonic acid from the sn-2 position of phospholipids via cytosolic phospholipase A2 for conversion to eicosanoids. For example, among the potent bioactive eicosanoids produced by leukocytes, the prostaglandins and leukotrienes are broadly considered pro-inflammatory mediators (50
). The sn-2 position of phospholipids that can become substituted with ω-3 fatty acids (DHA, EPA) is currently thought to simply compete for these enzymatic reactions, thus blocking the utilization of arachidonate and production of specific eicosanoids that are pro-inflammatory and pro-thrombotic mediators. This view is consistent with results from both cultured and isolated cells in vitro
when ω-3 fatty acids are supplied to isolated cells (41
). To address these points in a pathophysiologic setting in the present investigations, we determined the appearance of EPA and DHA at local sites of inflammation in exudates from circulation.
By monitoring both deuterium labeled d5
-EPA and d5
-DHA levels from circulation as well as increases in protein levels within exudates, we found that they appear coincident in the forming exudates. Of interest, both d5
-EPA and d5
-DHA were identified in exudates within 1 h of initiation of inflammation and maintained levels up to 48 h. At 48 h, both d5
-EPA and d5
-DHA levels were significantly greater within the exudates than their levels at 24 h. The first peak of D5
fatty acids was directly delivered from the circulation. Meanwhile, the second peak at 48 h likely reflects recirculation and PLA2
expression in the resolution of inflammation. Moreover, cPLA2
were highly expressed during the resolution phase (51
). The second peak at 48 h could be from esterified d5
-EPA and d5
-DHA and released by PLA2
mechanisms. Since in human plasma the half-life of EPA is 67 h and that of DHA is 20 h (48
), the clearance of EPA and DHA is relatively slow compared to xenobiotic small molecules. Taken together, the present results suggest that circulating ω-3 fatty acids are available for sites of acute inflammation, initially, directly from circulation. The presence of albumin at sites of inflammation, by definition, determines whether the inflammatory site is considered an inflammatory exudate or transudate (37
). The main protein component in the inflammatory exudates generated in the current zymosan initiated peritonitis is, indeed, serum albumin demonstrated by 2D-gel electrophoresis and proteomics (35
). Albumin is a well appreciated carrier protein of unesterified fatty acids and particularly DHA (52
Hence the present results indicate that circulating DHA and EPA are directly utilized by developing inflammatory exudates and do not require specialized mobilization from complex lipids or specific phospholipase activation to initially impact inflammation and its timely resolution. Both EPA and DHA appeared rapidly in exudates in their unesterified form coincident with edema generation and movement of circulating albumin and leukocyte trafficking into the evolving inflammatory exudates. Lundy et al. also indicated that edema formation and time course of arachidonic acid in mouse peritonitis were parallel (53
). These findings in mice suggest that EPA and DHA are directly mobilized for resolvin production from the circulation via albumin as the most abundant and likely main carrier into the inflammatory sites. In humans, [13
C]-DHA in phosphatidylcholine was rapidly hydrolyzed and available as a free fatty acid in plasma (54
). Furthermore, after ingestion of supplement, non-esterified EPA is detected in plasma (55
). Taken together, these findings imply that in humans the circulating levels of EPA and DHA do not require storage and subsequent release from complex lipid precursors in order to have an important contribution to controlling inflammation and its resolution.
Next, we questioned the relationship between circulating ω-3 fatty acids and their anti-inflammatory properties in vivo
. Using a new microfluidic chamber approach to rapidly isolate human neutrophils directly from circulating whole blood via capture on P-selectin coated surface, we assessed the direct actions of both precursor DHA and RvD1 on single neutrophil chemotaxis responses. Earlier procedures required time-consuming isolation of neutrophils from whole blood prior to in vitro
analyses. These protocols involved several steps of centrifugation and red blood cell lysis that usually take several hours (56
) to perform that could lead to changes in the responsiveness and characteristics of the isolated neutrophils. By contrast, the P-selectin based capture of neutrophils from a single drop of whole blood (~5-10 μl) is capable of performing the PMN separation in less than 5 minutes. This short time interval is ideal for assessing the activation and /or inhibition status of neutrophils from the blood of healthy donors as well as patients. The combination of separation and assessment of shape and migration responses in the same chamber is closely akin to in vivo
scenarios where neutrophils roll on the endothelial surfaces (37
), stick to the endothelium in regions of higher selectin expression, and respond via chemotaxis in the gradient of a chemokine, e.g., IL-8, and migrate into tissues (see ref. (37)
). The small amount of blood used in the microfluidic chamber is an additional advantage for performing these analyses with human PMN because they circumvent the need for venous phlebotomy and associated risks.
Another key feature of this microfluidic chamber system is the ability to record real-time changes in morphology of PMN upon exposure to chemokines, DHA and lipid mediators such as RvD1, as well as to track migration through switches. The fast gradient switches in the chamber allowed visual assessment and recording of the earliest events after exposure of cells to RvD1 or native DHA as well as precise measurement of these change in migration direction and velocity. Currently, no other chemotaxis systems are available that allow this level of precision or time resolution. For instance, in Boyden chambers (57
), one cannot directly observe the cells, the information obtained is indirect, and the system requires a large number of cells and separate controls. The Boyden chamber is an “end point assay” and one would not be able to assess whether neutrophils migrated before or after the addition of the inhibitor, or by adding the inhibitor before cells start moving if the effect would extrapolate to chemotaxis. In the Dunn and Zigmond chambers (58
), although one could visualize the cells in real time, it is not possible to swiftly switch between cell incubation and exposure conditions as with the present microfluidics chambers. The engineered microstructured valves for switching between independent gradients allowed the same neutrophils to serve as positive controls for migration in a chemoattractant gradient and then probed with either RvD1 or its precursor native DHA. The preservation of chemoattractant gradient before and after the switch is important for avoiding neutrophil responses to sudden changes of chemoattractant gradient. Of interest, sudden decrements in the concentration of chemokines alone have the potential to stop neutrophil migration for 3-5 minutes (60
). Thus, the present direct assessment of DHA with PMN indicates that DHA itself is not a potent bioactive `stop signal' for PMN but rather requires exudate conversion to RvD1 to evoke its signaling properties on these cells. Hence, following their actions, local tissues inactivate resolvins and permit organs to return to homeostasis.
Along these lines, ischemia-reperfusion is an event of significant clinical importance. Reperfusion related tissue injury occurs during surgical procedures, particularly those involving extremities, causing both local and remote organ injury as well as increasing costs associated with prolonged post-operative recovery (38
). Briefly, when vessels are surgically clamped or occlude, stasis of blood at the occlusion site leads to local ischemia and the neutrophils in that blood become activated. Upon release of the occlusion, activated leukocytes give rise to second/or remote organ injury. For example, when neutrophils from the occluded vessels of the hind limb reach the lung they cause local tissue damage (31
). In many respects, the aberrant activation of neutrophils in this scenario exhibits features similar to those in uncontrolled acute inflammation and neutrophil mediated tissue damage (31
). Given the clinical importance and patho-physiology of this type of organ injury, we investigated the direct actions of DHA, resolvins and related stable analogs (i.e., directly comparing the actions of RvD1, its 17-(R/S
)-methyl analog, RvE1, and its 19-p
-fluorophenoxy analog) in ischemia-reperfusion second organ injury. At equivalent doses, DHA was not protective while RvD1 and its analog as well as the stable analog of RvE1 showed potent anti-leukocyte actions each reducing infiltration into lung tissues.
Native RvE1 itself was not able to protect the lung at these low doses likely because of local inactivation. Of interest, RvE1 itself is both anti-inflammatory and pro-resolving in several inflammatory disease models (45
). Recently, RvE1 was also shown to have potent actions in preventing joint damage and cartilage destruction in collagen-induced rodent arthritis*
and protects from inflammation induced bone loss in periodontal disease (39
). Both RvD1 and RvE1 undergo site specific metabolic inactivation (26
). Thus, the RvD1 and RvE1 analogs that display potent organ protective actions as demonstrated herein () may provide new approaches to reduce organ damage characterized by excessive PMN infiltration. PMN are now appreciated to play an important role in the pathogenesis of many life-long chronic inflammatory diseases such as arthritis (61
). Recurring bouts of unresolved local acute inflammation may underlie many chronic inflammatory diseases. Thus, new means of monitoring neutrophils from peripheral whole blood within minutes, as demonstrated here with these newly designed microfluidic chambers may have implications in selecting appropriate therapies for acute and chronic inflammatory diseases.
In summation, we tracked the movements of labeled EPA and DHA from circulation to inflammatory exudates and recorded in real time the direct actions of RvD1 on human PMN migration using a newly engineered microfluidic chamber (32
). Using this approach, we visualized individual cellular responses with neutrophils obtained from a single drop of peripheral blood in less than 5 min. This new system provided a unique opportunity to investigate the actions of RvD1 at the single cell level. As shown in the supplementary video (online)
, PMN moving along the chemotactic gradient stopped movement essentially immediately after exposure to RvD1 but not with equimolar amounts of its exudate precursor DHA.
We also demonstrate new organ protective actions of resolvins in a murine model of second organ lung injury following hind limb ischemia-reperfusion. These results emphasize the substantial contribution of unesterified ω-3 fatty acids levels in circulation and their rapid availability via edema formation in exudates to mount resolution and assist in the return of local tissues to homeostasis. Recent review of the evidence for beneficial effects of fatty acids in human inflammation indicates a poor relationship and predictive value suggesting specific dose response relationships for dietary ω-3 fatty acids, and fatty acid profiles of immune cells differ between species (62
). These are consistent with our results and suggest that circulating ω-3 DHA and EPA levels and their transfer by edema rather than phospholipid esterified precursors are an initial direct source for exudate biosynthesis of resolvins, and thus should be taken into account in evaluating the impact of nutrition in humans. The new pathways uncovered here are likely to be relevant in maintaining health as well as in the many diseases characterized by excessive uncontrolled inflammation.